In animals and advanced microorganisms, the enzymes involved in synthesis of fatty acids de novo are integrated into a fatty acid synthetase multienzyme complex. We have found that the component activities are linked together on two polyfunctional polypeptides. Recent results described in our progress report indicate the two polyfunctional polypeptides are identical. Using limited trypsinization, we have detached, in active from, the two thioesterase I domains from the fatty acid synthetase homodimer. The thioesterase I activity is responsible for termination of growth of the acyl chain at 16 C atoms on the multienzyme. Whereas the isolated fatty acid synthetases from animal tissues synthesize mainly C16, in the lactating mammary gland the product specificity of the multienzyme is modified by a second component, a mammary gland specific enzyme, thioesterase II. The mechanism of interaction of the two enzymes is under investigation. The properties of thioesterase I and II are being studied from the aspect of structure, specificity and amino acid sequence around the active site, to establish a basis for their different functions.